M-MLV Neoscript Reverse Transcriptase
Neoscript Reverse Transcriptase wani juzu'i ne da aka samu ta hanyar tantance maye gurbin kwayar halittar M-MLV na asalin cutar sankarar bargo na Moloney murine da kuma magana a cikin E.coli.Enzyme yana kawar da ayyukan RNase H, yana da mafi girman jurewar zafin jiki, kuma ya dace da jujjuya juzu'i mai zafi.Sabili da haka, yana da taimako don kawar da mummunan tasirin babban tsari na RNA da abubuwan da ba na musamman ba akan haɗin cDNA, kuma yana da kwanciyar hankali mafi girma da kuma juyar da ikon rubutun rubutun.Enzyme yana da kwanciyar hankali mafi girma kuma yana jujjuya ƙarfin rubutun rubutu.
Abubuwan da aka gyara
1.200 U/μL Neoscript Reverse Transcriptase
2.5 × Maɓalli na Farko (na zaɓi)
* 5 × First-Strand Buffer bashi da dNTP, da fatan za a ƙara dNTPs lokacin shirya tsarin amsawa
Aikace-aikacen da aka ba da shawarar
1.Mataki ɗaya qRT-PCR.
2.gano kwayar cutar RNA.
Yanayin Ajiya
-20 ° C don ajiya na dogon lokaci, ya kamata a gauraye da kyau kafin amfani, kauce wa daskare-narke akai-akai.
Ma'anar Naúrar
Raka'a ɗaya ta haɗa 1 nmol na dTTP a cikin mintuna 10 a 37°C ta amfani da poly(A)•oligo(dT)25as template/primer.
Kula da inganci
1.SDS-PAGE tsarki na electrophoretic fiye da 98%.
2.Amplification hankali, sarrafa tsari-zuwa-tsari, kwanciyar hankali.
3.Babu wani aiki na nuclease, babu exogenous endonuclease ko exonuclease gurbatawa
Saitin Amsa don Maganin Amsa Sarkar Farko
1.Shiri na dauki cakuda
| Abubuwan da aka gyara | Ƙarar |
| Oligo(dT)12-18 Firamare ko Random Primera Ko Gene Specific Primersb | 50 pml |
| 50 pm (20-100 pm) | |
| 2 pml | |
| 10 mM dNTP | 1 μl |
| Samfura RNA | Jimlar RNA≤ 5μg;mRNA≤ 1 μg |
| RNase-free dH2O | ku 10 μl |
Bayanan kula:a/b: Da fatan za a zaɓi nau'ikan firamare daban-daban gwargwadon buƙatun gwajin ku.
2.Yi zafi a 65 ° C na minti 5 kuma a kwantar da sauri a kan kankara na 2mins.
3.Ƙara abubuwan da ke biyo baya zuwa tsarin da ke sama zuwa jimlar ƙarar 20µL kuma a haɗa su a hankali:
| Abubuwan da aka gyara | girma (μL) |
| 5 × Farko-Tsarin Buffer | 4 |
| Neoscript Reverse Transcriptase (200 U/μL) | 1 |
| Mai hana RNase (40 U/μL) | 1 |
| RNase-free dH2O | Ku 20 μl |
4. Da fatan za a yi martani bisa ga sharuɗɗa masu zuwa:
(1) Idan Random Primer da ake amfani, da dauki ya kamata a da za'ayi a 25 ℃ for 10mins, sa'an nan a 50 ℃ for 30 ~ 60mins;
(2) Idan aka yi amfani da Oligo dT ko takamaiman abubuwan da ake amfani da su, yakamata a aiwatar da dauki a 50 ℃ na 30 ~ 60mins.
5.Yi zafi a 95 ℃ na 5mins don kunna Neoscript Reverse Transcriptase da kuma dakatar da amsawa.
6.Za'a iya amfani da samfuran juzu'i kai tsaye a cikin amsawar PCR da ƙimar ƙimar PCR mai haske, ko adana su a -20 ℃ na dogon lokaci.
PCR Raiki:
1.Shiri na dauki cakuda
| Abubuwan da aka gyara | Hankali |
| 10 × PCR Buffer (kyauta dNTP, Mg²+ kyauta) | 1× |
| dNTPs (10mM kowane dNTP) | 200 μM |
| 25 mMgCl2 | 1-4 mm |
| Taq DNA Polymerase (5U/μL) | 2-2.5 U |
| Fitowa 1 (10 μM) | 0.2-1 m |
| Fitowa 2 (10 μM) | 0.2-1 m |
| Samfuraa | ≤10% Maganin Amsa Sarkar Farko (2 μL) |
| ddH2O | ku 50 μl |
Bayanan kula:a: Idan an ƙara maganin sarkar farko da yawa, ana iya hana aikin PCR.
2.Hanyar Amsa PCR
| Mataki | Zazzabi | Lokaci | Zagaye |
| Pre-denaturation | 95 ℃ | 2-5 min | 1 |
| Denaturation | 95 ℃ | 10-20 seconds | 30-40 |
| Annealing | 50-60 ℃ | 10-30 seconds | |
| Tsawaita | 72 ℃ | 10-60 seconds |
Bayanan kula
1.Ya dace don inganta yanayin juzu'i a cikin kewayon 42 ℃ ~ 55 ℃.
2.Yana da mafi kyawun kwanciyar hankali, ya dace da haɓaka jujjuyawar zafin jiki mai girma.Bugu da ƙari, yana da kyau don wucewa ta hanyar rikitattun yankuna na RNA.Hakanan, shiya dace don gano ƙimar RT-PCR-mataki-mataki-ɗaya multix fluorescence.
3.Kyakkyawan dacewa tare da nau'ikan haɓaka enzymes na PCR kuma ya dace da halayen halayen RT-PCR mai girma.
4.Ya dace da babban azanci na mataki-ɗayan haske mai ƙididdige amsawar RT-PCR, yadda ya kamata ya inganta ƙimar gano ƙarancin ƙima na samfuri.
5.Ya dace da ginin ɗakin karatu na cDNA.
